A kit for microbial detection and its application

Abstract

The present invention discloses a kit for microbial detection and its application, belonging to the field of immunoassay technology. The kit for microbial detection comprises an enzyme label plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing solution, a color developing solution and a stop solution; the capture antibody and the HRP-labeled antibody are antibodies that specifically bind to human Blastocystis antigens. The kit for microbial detection provided by the present invention can detect the content of human Blastocystis in feces, is accurate, sensitive, simple and easy to use, overcomes the defects of requiring professionals and equipment for microscopic screening of human Blastocystis, and is easy to miss, and the operation can be completed in an ordinary laboratory, and has good promotion.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a kit for microbial detection and application thereof. Background Art

[0002] Blastocystis is a common opportunistic intestinal parasite. It is a single-cell protozoan that is widely distributed throughout the world. It mainly parasitizes the large intestine of humans, other primates, dogs, pigs, cats, mice, rats, rabbits, guinea pigs, frogs, snakes and other animals. Among them, Blastocystis hominis is the human-derived species of Blastocystis. Blastocystis hominis has various morphologies, including vacuolar, granular, amoeboid, multifission, and cystic types. The size varies greatly, with a diameter range of 2-200μm.

[0003] Blastocystis hominis is transmitted through cysts excreted in feces, and the transmission route is fecal-oral transmission. Any patient, carrier or host that excretes Blastocystis hominis in feces can become a source of infection. Improper fecal management allows Blastocystis hominis to be transmitted through water, food and utensils. Most infected people have no clinical specific manifestations, and the Blastocystis organisms only live in the human digestive tract without causing harm. However, when the human immune system declines, opportunistic pathogens such as Blastocystis hominis take advantage of the situation and gradually become dominant pathogens, causing abdominal pain, diarrhea, abdominal distension, anorexia, nausea, vomiting and other symptoms. Some severe patients may suffer from severe diarrhea, hypoproteinemia, generalized edema, etc., and even endanger their lives. Combined with the aging population in today's society, the increase in diseases such as tumors, AIDS, and tuberculosis, and the widespread use of drugs such as immunosuppressants, the number of people with low immunity continues to increase. Therefore, the research and application of detection and diagnostic methods for Blastocystis hominis is particularly important.

[0004] The laboratory diagnosis of human Blastocystis is mainly through etiological examination, and the diagnosis can be confirmed by detecting the parasite in the specimen. However, due to the diverse morphology and large size differences of human Blastocystis, it is sensitive to oxygen, and the etiological detection method is easily interfered by other components in the feces. Therefore, it is difficult to identify and diagnose, which can easily lead to a certain proportion of missed detection and misdiagnosis. In addition to traditional etiological examinations, molecular biological diagnosis is currently one of the conventional diagnostic methods for human Blastocystis disease. Among them, PCR technology has high sensitivity and specificity, especially in interspecies differential diagnosis. However, PCR requires specialized equipment and technicians, and is usually completed in specialized testing centers, which limits its wide application in the field.

[0005] As an opportunistic intestinal parasite, Blastocystis hominis faces many challenges in diagnosis and prevention. In order to detect Blastocystis hominis more quickly, simply and accurately, it is necessary to continuously explore new diagnostic methods and technologies to meet clinical needs. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, one of the objects of the present invention is to provide a kit for microbial detection, which can be used to detect human Blastocystis, has easy sample acquisition, simple operation and wide application.

[0007] A second object of the present invention is to provide an application of the kit for microbial detection.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A kit for microorganism detection comprises an enzyme label plate coated with a capture antibody, an HRP labeled antibody, an antigen standard, a washing solution, a color developing solution and a stop solution.

[0010] Furthermore, the capture antibody and the HRP-labeled antibody are both antibodies that specifically bind to human Blastocystis antigens.

[0011] Furthermore, the capture antibody is the monoclonal antibody 4G5, the heavy chain amino acid sequence of the monoclonal antibody 4G5 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0012] Furthermore, the HRP-labeled antibody is prepared from the monoclonal antibody 2E11, the heavy chain amino acid sequence of the monoclonal antibody 2E11 is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.6.

[0013] Furthermore, the antigen standard is a Blastocystis hominis antigen standard; the washing solution is PBST; the color developing solution is TMB; and the stop solution is 2M sulfuric acid.

[0014] Furthermore, the antibody that specifically binds to human Blastocystis antigen is prepared by the following method:

[0015] (1) Isolate human Blastocystis from feces, freeze-thaw repeatedly, ultrasonically disrupt and centrifuge to prepare human Blastocystis antigen;

[0016] (2) immunizing mice with the human Blastocystis antigen in step (1), fusing spleen cells of the immunized mice with myeloma cells to form hybridoma cells;

[0017] (3) ELISA was used to screen tumor cells that secrete human Blastocystis monoclonal antibodies, and monoclonal antibodies 4G5 and 2E11 were obtained.

[0018] Furthermore, the step of coating the ELISA plate with the capture antibody is as follows: diluting the monoclonal antibody 4G5 with the antibody coating solution and adding it to the microwells of the ELISA plate, sealing the plate with a sealing film, placing it at 4°C for coating overnight, adding the blocking solution and blocking it at room temperature for a period of time, discarding the liquid in the wells of the ELISA plate, drying and sealing, and storing it at 4°C.

[0019] Furthermore, the preparation method of the HRP labeled antibody is as follows: monoclonal antibody 2E11 is added to HRP solution oxidized by NaIO4, reacted at room temperature in the dark, NaBH4 is added and reacted at 4°C for a period of time, and dialyzed against PBS overnight.

[0020] Furthermore, the step of oxidizing HRP with NaIO4 is as follows: 0.2 M NaIO4 solution is mixed with 40 mg / mL HRP solution, and dialyzed in 10 mM NaAc buffer at 4°C overnight.

[0021] The second object of the present invention is achieved by adopting the following technical solution:

[0022] The kit for microbial detection is used for detecting the content of human Blastocystis in feces.

[0023] Furthermore, the detection steps are:

[0024] (1) After pretreatment of the sample to be tested, repeated freezing and thawing, ultrasonic disruption and centrifugation were performed to obtain the supernatant;

[0025] (2) Take out the capture antibody-coated ELISA plate, return it to room temperature, wash the plate with PBST washing buffer and spin dry; add the sample treated in step (1), incubate in a room temperature incubator and then wash the plate;

[0026] (3) Add the HRP-labeled antibody to the system in step (2), incubate at room temperature for a period of time, wash the plate with PBST and spin dry;

[0027] (4) adding a color developing solution to the system after the reaction in step (3), developing the color for a period of time at room temperature in the dark, and then adding a stop solution to terminate the reaction;

[0028] (5) Use an ELISA reader for dual-wavelength detection. Subtract the OD value at 630 nm from the OD value at 450 nm and calculate the result using the standard curve.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The kit for microbial detection provided by the present invention can detect the content of human Blastocystis in feces, is accurate, sensitive, simple and easy to use, overcomes the defects of microscopic screening of human Blastocystis requiring professionals and equipment, and easy to miss detection, and can be completed in an ordinary laboratory, and has good popularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The principle of the kit for microbial detection of the present invention; wherein ① is an ELISA plate coated with a capture antibody; ② is the addition of a sample to be tested; ③ is the washing solution after the plate is washed, leaving only the human Blastocystis antigen bound to the capture antibody in the ELISA plate; ④ is the HRP-labeled antibody bound to the antigen;

[0032] Figure 2 This is the standard curve of the kit for microbial detection of the present invention. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0034] Example 1

[0035] Preparation of monoclonal antibodies against Blastocystis hominis:

[0036] Take 1g of stool sample from a patient with diarrhea, dilute with 10 mL of saline, shake and mix on an oscillator, centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, add 10 mL of saline to the precipitate, shake and mix on an oscillator, centrifuge again at 1000 rpm for 5 min, discard the supernatant, add 10 mL of saline to the precipitate and mix. Take 0.5 mL of the stool solution and inoculate it into LES biphasic culture medium, and subculture continuously for 2 weeks. Then inoculate the above cultured protozoa into soft agar IMDM solid phase culture medium (containing 15% newborn calf serum, 0.3% agar, 100 U / mL penicillin, 100 µg / mL streptomycin and 2.5 µg / mL amphotericin). After culturing for 4 weeks, when a mass-like protozoan colony appears, pick a single mass-like protozoan colony with an inoculation loop and inoculate it into IMDM liquid phase culture medium (containing 15% newborn calf serum, 100 U / mL penicillin, 100 µg / mL streptomycin and 2.5 µg / mL amphotericin). Continue culturing for 3 weeks. Take 400 μL of the above culture mixture for sequencing. The sequencing primers are:

[0037] Upstream primer: 5' TCATACGCTCGTCTCAAA 3' (SEQ ID NO. 1);

[0038] Downstream primer: 5' TGATAGGGCAGAAACTTGA 3' (SEQ ID NO. 2);

[0039] The sequencing results were compared with the Blastocystis hominis genome data on the NCBI website to identify whether the isolated human Blastocystis was correct.

[0040] The isolated human Blastocystis was cultured anaerobically in a 37°C incubator using liquid IMDM medium for 7 days. The human Blastocystis was resuspended in sterile PBS, frozen and thawed three times, and then ultrasonically broken in an ice bath for 20 min. The supernatant was centrifuged at 10,000 rpm for 10 min, and the supernatant was obtained, which was the human Blastocystis antigen. The protein concentration was determined by the bicinchoninic acid (BCA) method, and the concentration of human Blastocystis antigen was adjusted to 5 μg / mL, and protease inhibitors were added and stored at -80°C for later use.

[0041] Balb / c female mice were immunized three times by subcutaneous injection at the abdomen. The antibody titer of mouse plasma was determined by indirect ELISA, and the mice with the highest titer were selected for hybridoma cell fusion. The cell number ratio of spleen cells to myeloma cells SP2 / 0 was 10:1, the fusion time was 2 minutes, and 50% polyethylene glycol was used as the fusion reagent. After about 10 days of screening and cultivation in HAT screening medium, the supernatant was taken for ELISA detection to screen out positive cell lines with better performance, and the positive cells were subcloned by limiting dilution method. 1×10 7 The subcloned cells were intraperitoneally injected into Balb / c mice. On the 13th day, the mouse ascites was collected and filtered with a 0.2μm filter. The ascites was purified by ion exchange chromatography, then added to a concentration column, centrifuged at 5000rpm for 15min, and PBS was added for replacement to obtain the concentrated monoclonal antibody. The antibody concentration was adjusted to 10mg / mL for later use.

[0042] The two most active monoclonal antibodies were named 2E11 and 4G5, and their variable region sequences were determined. Monoclonal antibody 2E11 and monoclonal antibody 4G5 include a light chain and a heavy chain, respectively; wherein the heavy chain amino acid sequence of monoclonal antibody 2E11 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4; the heavy chain amino acid sequence of monoclonal antibody 4G5 is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.6.

[0043] SEQ ID NO.3

[0044] EVKLDQSGADLVKPGGTAKLSMVVSGFGESSFNTREWARSTPDRRLETAAGRTDVSYYNDNEGGKGRFSVTRETVKTTAYLNCSSSRSEENLAYYCVIGDAIMYVQWGQGTAVTVSA;

[0045] SEQ ID NO.4

[0046] QTVVNTPMALLGSLASQLSANSDLADREWFRQHMATVLKSLAYFASDFDGACDHSSGSTNATNGTLIASILNAEDDGDYYCCKSNLTSISQDTNSSNWFATGTRLEI;

[0047] SEQ ID NO.5

[0048] QVQLNQSGMELLHPGVNVRISNKGSGYRFTSYCADLAKTSKLHSLDNIGGNPDSGNMQKKKNDYRYKATPNAEQSSSNGFMEAARLTSDENGIYYCSRLAGNYGDYWGQGTGLLVSS;

[0049] SEQ ID NO.6

[0050] QAVGIQDTALTQTCGETVNVQCHTSVVQWVNDKPERLFNGLIAATYGAFMDGVPARFSGSIAADKAGVTINAGQTEDDVIYFCGLLYLVGSRNPWSMVFGAGTHLTVK。

[0051] Example 2

[0052] Kit for microbial detection and its application:

[0053] Preparation of HRP labeled antibody: The monoclonal antibody 2E11 prepared in Example 1 was dialyzed in 50mM carbonate buffer for 24h, during which the solution was changed twice. After dialysis, its concentration was adjusted to 2mg / mL. Weigh 2mg HRP dry powder and dissolve it in 100μL ddH2O. Weigh 21mg NaIO4 and dissolve it in 1 mL ddH2O to prepare a 0.2 M NaIO4 solution. Mix 0.4mL NaIO4 solution with 2mL HRP solution, keep it at room temperature and away from light for 40min, and immerse it in 10 mM NaAc buffer and dialyze it overnight at 4℃. Mix equal volumes of HRP solution and monoclonal antibody 2E11, and stir gently at room temperature and away from light for 3 hours. Weigh 0.4mg NaBH4, dissolve it in 20μL ddH2O, add all of it to the reaction solution in the previous step, mix it, place it at 4℃ for reaction for 2 hours, and then dialyze it in PBS at 4℃ overnight.

[0054] Preparation of capture antibody-coated ELISA plate: Select the monoclonal antibody 4G5 prepared in Example 2 as the capture antibody. Use an ELISA 96-well plate as a solid phase carrier, dilute the capture antibody to 2 μg / mL with antibody coating solution, add 100 μL / well to the microwells of the ELISA plate, seal the plate with a sealing film and place it at 4°C for overnight coating. Discard the liquid in the wells, wash the plate 3 times with ELISA washing solution, add 250 μL / well of blocking solution, and place it at room temperature for 2 hours. Discard the liquid in the plate wells, dry for 16 hours, seal, and store at 4°C.

[0055] Use of the kit: The principle of the kit for detecting microorganisms by the double antibody sandwich ELISA method is as follows Figure 1 As shown in the figure, ① is the ELISA plate coated with the capture antibody; ② is the addition of the sample to be tested; ③ is the washing solution after the plate is washed, and only the human Blastocystis antigen bound to the capture antibody remains in the ELISA plate; ④ is the HRP-labeled antibody bound to the antigen, and the HRP enzyme coupled to the HRP-labeled antibody can be used to detect the content of human Blastocystis in the sample to be tested.

[0056] Standard curve drawing: 40 min before the experiment, remove the capture antibody-coated ELISA plate, return to room temperature, wash the plate 3 times with PBST washing buffer and spin dry. Add 100 μL of different dilutions of human Blastocystis antigen (80ng / mL, 60ng / mL, 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL) and set up a blank control, incubate in a room temperature incubator for 2h, wash the plate 3 times and spin dry. Add 100 μL of HRP-labeled antibody working solution to the reaction well, incubate in a room temperature incubator for 40 min, wash the plate 3 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, color at room temperature in the dark for 10min, and add 70 μL of 2M sulfuric acid to terminate the reaction. Use an ELISA reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm. Draw a standard curve with the concentration of the standard as the horizontal axis (ng / mL) and OD 450 - OD 630 as the vertical axis. The results are shown in Figure 1. Figure 2 As shown, the detection range of the kit for microbial detection in this application is 2.5-60 ng / mL, R 2 = 0.9993.

[0057] Test Example 1

[0058] Double Antibody Sandwich ELISA Kit Specificity:

[0059] Common intestinal microorganisms in feces, including ① Lactobacillus, ② Staphylococcus, ③ Streptococcus, ④ Prevotella, ⑤ Bacteroides fragilis, ⑥ Clostridium, ⑦ Clostridium difficile, ⑧ Bifidobacterium catenulatum, ⑨ Corynebacterium, and ⑩ Propionibacterium, were collected by centrifugation after cultivation, and resuspended in sterile PBS, and then frozen and thawed three times, and then ultrasonically broken in an ice bath for 20 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected, which was the antigen of intestinal microorganisms. The concentration was determined by the BCA method, adjusted to 20 ng / mL, and protease inhibitors were added and stored at -80°C.

[0060] 30 min before the experiment, remove the capture antibody-coated ELISA plate, return to room temperature, wash the plate three times with PBST washing buffer and spin dry. Add 100 μL of 20 ng / mL human Blastocystis antigen or intestinal microbial antigen (called bacteria ①-⑩) 4 copies each, incubate in a room temperature incubator for 2 hours, wash the plate three times and spin dry. Add 100 μL of HRP-labeled antibody working solution to the reaction well, incubate in a room temperature incubator for 40 min, wash the plate three times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, color at room temperature in the dark for 10 min, and add 70 μL of 2M sulfuric acid to terminate the reaction. Use an ELISA reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the results using the standard curve. The results showed that the antibody did not react with other similar proteins (Table 1).

[0061] Table 1

[0062]

[0063] Test Example 2

[0064] Stability of double antibody sandwich ELISA kit:

[0065] Place the capture antibody-coated ELISA plate, HRP-labeled antibody, and human Blastocystis antigen standard at 37°C to test its stability for 20 days, and perform human Blastocystis antigen detection every 5 days. Place the capture antibody-coated ELISA plate, HRP-labeled antibody, and human Blastocystis antigen standard in a 4°C refrigerator for 2 years, and take them out every 12 months for human Blastocystis antigen detection.

[0066] The detection method is as follows: take the capture antibody-coated ELISA plate, wash the plate three times with PBST washing buffer and spin dry. Add 100 μL of different dilutions of human Blastocystis antigen (60 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL) and set up a blank control, incubate in a room temperature incubator for 2 hours, wash the plate three times and spin dry. Add 100 μL of HRP-labeled antibody working solution to the reaction well, incubate in a room temperature incubator for 40 min, wash the plate three times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, color at room temperature in the dark for 10 min, and add 70 μL of 2M sulfuric acid to terminate the reaction. Use an ELISA reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm. The standard curve was drawn with the concentration of the standard as the horizontal axis (ng / mL) and OD 450 - OD 630 as the vertical axis. The results are shown in Table 2. When the kit was exposed at 37°C for 15 days and stored at low temperature for 2 years, the OD value gradient of the standard curve was good, indicating that the kit had good stability.

[0067] Table 2

[0068]

[0069] Test Example 3

[0070] Blastocystis hominis test:

[0071] Take 1g of fecal samples from 20 volunteers who have experienced diarrhea within a week, dilute with 10 mL of saline, shake and mix on an oscillator, centrifuge at 1000 g for 5 min at room temperature, discard the supernatant, add 10 mL of saline to the precipitate, shake and mix on an oscillator, centrifuge again at 1000 g for 5 min, discard the supernatant, add 10 mL of saline to the precipitate and mix. After repeated freezing and thawing 3 times, ultrasonically break the sample in an ice bath for 20 min, centrifuge at 10000 rpm for 10 min, and take the supernatant. The samples are recorded as fecal samples 1-20. Tap water was taken as a negative control, and 10 ng / mL of human Blastocystis antigen was used as a positive control.

[0072] 30 min before the experiment, remove the capture antibody-coated ELISA plate, return to room temperature, wash the plate three times with PBST washing buffer and spin dry. Add 100 μL of the sample to be tested (each sample is repeated three times), incubate in a room temperature incubator for 2 hours, wash the plate three times and spin dry. Add 100 μL of HRP-labeled antibody working solution to the reaction well, incubate in a room temperature incubator for 40 min, wash the plate three times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, color at room temperature in the dark for 10 min, and add 70 μL of 2M sulfuric acid to terminate the reaction. Use an ELISA reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm, and calculate the results using the standard curve. As shown in Table 3, the stool samples of 8 volunteers were tested positive for human Blastocystis antigen, and the detection concentrations were all within the linear range of the kit of the present invention, indicating that the detection method established by the present invention can use stool as a sample to complete the detection of human Blastocystis infection.

[0073] Table 3

[0074]

[0075] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A kit for microbial detection, characterized in that: The kit comprises an ELISA plate coated with a capture antibody, an HRP labeled antibody, an antigen standard, a washing solution, a color developing solution and a stop solution; The capture antibody and the HRP-labeled antibody are both antibodies that specifically bind to human Blastocystis antigens; The antibody specifically binding to human Blastocystis antigen is prepared by the following method: (1) Isolate human Blastocystis from feces, freeze-thaw repeatedly, ultrasonically disrupt and centrifuge to prepare human Blastocystis antigen; (2) immunizing mice with the human Blastocystis antigen in step (1), fusing spleen cells of the immunized mice with myeloma cells to form hybridoma cells; (3) ELISA was used to screen tumor cells that secrete human Blastocystis monoclonal antibodies, and monoclonal antibodies 4G5 and 2E11 were obtained; The capture antibody is monoclonal antibody 4G5, the heavy chain amino acid sequence of the monoclonal antibody 4G5 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4; The HRP labeled antibody is prepared from the monoclonal antibody 2E11, the heavy chain amino acid sequence of the monoclonal antibody 2E11 is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.6; The antigen standard is a Blastocystis hominis antigen standard; the washing solution is PBST; the color developing solution is TMB; and the stop solution is 2M sulfuric acid.

2. The kit for microbial detection according to claim 1, characterized in that: The steps of coating the ELISA plate with the capture antibody are as follows: diluting the monoclonal antibody 4G5 with the antibody coating solution and adding it to the microwells of the ELISA plate, sealing the plate with a sealing film, placing it at 4°C for coating overnight, adding the blocking solution and blocking it at room temperature for a period of time, discarding the liquid in the wells of the ELISA plate, drying and sealing, and storing it at 4°C.

3. The kit for microbial detection according to claim 1, characterized in that: The preparation method of the HRP labeled antibody is as follows: monoclonal antibody 2E11 is added to HRP solution oxidized by NaIO4, reacted at room temperature in the dark, NaBH4 is added, the reaction is continued at 4°C for a period of time, and dialyzed against PBS overnight.

4. The kit for microbial detection according to claim 3, characterized in that: The steps of oxidizing HRP with NaIO4 are as follows: 0.2 M NaIO4 solution is mixed with 40 mg / mL HRP solution, and dialyzed in 10 mM NaAc buffer at 4°C overnight.

5. The use of the kit for microbial detection according to claim 1, characterized in that: The kit is used to detect the content of Blastocystis hominis in feces.

6. Use of the kit for microbial detection according to claim 5, characterized in that: The detection steps are: a. Take the sample to be tested and pre-treat it, freeze-thaw it repeatedly, and ultrasonically disrupt it, then centrifuge it and take the supernatant; b. Take out the capture antibody-coated ELISA plate, return it to room temperature, wash the plate with PBST washing buffer and spin dry, add the sample treated in step a, incubate in a room temperature incubator and then wash the plate; c. Add the HRP-labeled antibody to the system in step b, incubate at room temperature for a period of time, wash the plate with PBST and spin dry; d. Add the color developing solution to the system after the reaction in step c, develop the color at room temperature in the dark for a period of time, and then add the stop solution to terminate the reaction; e. Use a microplate reader for dual-wavelength detection, subtract the OD value at 630 nm from the OD value at 450 nm, and calculate the result using the standard curve.

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